Natriuretic peptides (NPs) are known for their role in cardiovascular homeostasis, diuresis, natriuresis and vasodilation. They act in the body to oppose the activity of the renin-angiotensin system and enhance excretion of sodium and water. There are four known types of natriuretic peptides in humans: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), S-type natriuretic peptide (CNP) and dendroaspis natriuretic peptide (DNP). Isoforms of these occur in nature across species.
ANP is secreted by atrial myocytes in response to increased intravascular volume. Once it is in the circulation, its effects are primarily on the kidney, vascular tissue and adrenal gland, in which its actions lead to the excretion of sodium and water by the kidneys and a decrease in intravascular volume and blood pressure (Atlas et al., 1987, In Atrial Hormones and Other Natriuretic Factors, P. J. Mulrow et al., Eds, Am. Physiol. Soc., Bethesda, Md., pp. 53-76).
BNP is of myocardial cell origin and, like ANP, circulates in human plasma (de Bold et al., 1981, Life Science, 28(1): 89-94; Burnett et al., 1986, Science, 231(4742): 1145-1147). BNP is natriuretic, renin-inhibiting, vasodilating and lusitropic (Mukoyama et al., 1991, J. Clin. Invest., 87(4): 1402-1412; Yamamoto et al., 1996, Am. J. Physiol., 271(6 Pt 2):R1529-1534; Grantham et al., 1997, Natriuretic Peptides in Health and Disease, Samson W. K. 11e.r., eds, Humana Press, pp. 309-326).
CNP is of endothelial cell origin and functions as a vasodilating and growth-inhibiting peptide (Suga et al., 1992, J. Clin. Invest., 90(3): 1145-1149; Stingo et al., 1992, Am. J. Physiol., 262(1 Pt 2):H308-312; Koller et al., 1991, Science, 252(5002): 120-123).
DNP possesses structural similarity to ANP, BNP and CNP. It was originally found in the venom of Dendroaspis angusticeps and has been shown to vasorelax rodent aorta and isolated canine coronary arteries with potency comparable to that of ANP (Schweitz et al., 1992, J. Biol. Chem., 267: 13928-13932; Wenberg et al., 1997, Am. Coll. Cardiol., 29: 305a). Recently DNP immunoreactivity has been demonstrated in human circulation, and its plasma levels are elevated in congestive heart failure (CHF) (Schirger et al., 1999, Mayo Clin. Proc., 74: 126-130). A NP-like peptide, Lebetin 2 isoform alpha, was found in the venom of Vipera lepitera (Barbouche et al., 1996, FEBS Lett. 392:6-10). This peptide contains the consensus natriuretic peptide signature which indicates that it belongs to the same general family. Illustrative examples of known natriuretic peptides are given in Table 1.
Natriuretic peptides effect their biological role through three receptors: NPR-A, NPR-B and NPR-C. NPR-A and NPR-B have cytoplasmic guanylyl cyclase domains, which are activated upon ligand binding and lead to accumulation of intracellular cGMP. The tissue distribution of each receptor is different. While NPR-A is expressed in vasculature, kidney and adrenal glands, NPR-B is mainly expressed in the brain. NPR-C, is devoid of the kinase and cytoplasmic GC domains and is generally considered to be a clearance receptor for removing natriuretic peptides from the circulation, although other biological functions for it have been postulated (Murthy et al., 1999, J. Biol. Chem., 274(25): 17587-17592). U.S. Pat. Nos. 5,846,932 and 6,028,055 disclose potent ANP and BNP variants having decreased affinity for the human clearance or C-receptor (NPR-C). DNP has been shown to produce relaxation through both the NPR-A and NPR-C receptors.
ANP and BNP each bind to both NPR-A and NPR-C with high affinity, but only bind to NPR-B with low affinity, whereas CNP binds to NPR-B and NPR-C, but not appreciably to NPR-A (Potter L R and Hunter T, 2001, J. Biol. Chem. 276: 6057-6060). It is generally considered that the beneficial effects (natriuresis/diuresis or effects on renin/angiotensin system) of natriuretic peptides occur via the synthesis of the intracellular messenger cyclic-GMP (cGMP). NPRA is thought to be the primary ANP/BNP signaling molecule and has been suggested as the principal mediator of natriuretic peptide activities. Conclusive studies have determined that NPR-A is the only NP binding receptor that regulates natriuresis/diuresis and regulates the renin/angiotensin system (Kishimoto I, et al., 1996, Proc Natl Acad Sci USA 93: 6215-6219). In this study it was demonstrated that ANP does not bind to tissues from animals that are homozygous null mutants for NPR-A. These animals do not undergo natriuresis or diuresis in response to ANP and their aortic rings do not relax in response to that peptide. Shi et al. (2003, AJP—Renal 285:694-702) established that NPRA is critical in mediating the natriuresis, diuresis, and renal hemodynamic responses to acute blood volume expansion. From these studies it is apparent that, for a NP peptide to be of use in the treatment of CHF, it must act at the NPR-A receptor.
The ability for a NP to bind human NPR-A cannot be predicted from an analysis of primary sequence alone, nor can analysis of primary sequence predict the affinity with which a NP can bind to NPR-A. The sequences of both the hormones and their receptors vary across species. A consensus NP sequence has been identified for NPs but this is loosely defined and provides no information on whether the peptide acts as an NP, and no guidance as to whether the peptide is active at NPR-A in particular (and hence potentially useful for treating CHF), NPR-B and/or NPR-C. Schweitz et al. (1992 J. Biol. Chem. 267: 13928-13932) identified DNP as a ligand for NPR-A on the basis that the peptide relaxes aortic rings. In an early attempt to define features important for NPR-A binding, they proposed a conservative tripeptide sequence in the C-terminal part of the as then known natriuretic peptides consisting of (i) a small non-hydrophilic residue; (ii) a large hydrophobic residue and (iii) an arginine residue, as highlighted in FIG. 2. However, as is evident in later studies and in the present specification, this generalization does not hold and provides little guidance as to whether a natriuretic peptide has NPR-A activity, or otherwise.
ANP, BNP, CNP and DNP are synthesized from large precursors and the mature, active peptides have a 17-amino acid ring structure formed by an intramolecular disulfide linkage. In the human peptides, ten of these amino acids are identical, whereas the N-terminal head and C-terminal tail vary in both length and composition (see Kambayashi et al., 1990, FEBS Lett, 259: 341-345; Tawaragi et al., 1991, Biochem. Biophys. Res. Commun. 175: 645-651).
Due to their diverse actions on both the cardiovascular system and the kidney, ANP, BNP, CNP and DNP and their analogues have been the subject of great interest for developing novel therapeutics. For example, reference may be made to Lewicki et al. (U.S. Pat. Nos. 5,114,923, 4,804,650 and 4,757,048), Johnson et al. (U.S. Pat. No. 5,047,397), Johnson et al. (U.S. Pat. No. 4,935,492), and Wei et al. (U.S. Pat. No. 5,583,108). U.S. Pat. No. 5,583,108 discloses a chimera of ANP and CNP, termed vasonatrin peptide (VNP). VNP, which includes 22 amino acids of CNP and the 5 amino acids at the carboxy-terminus of ANP, has arterial and venous vasodilating and natriuretic effects. U.S. Pat. No. 6,407,211 discloses chimeric compounds comprising the C-terminal tail of DNP with the core ring structure of BNP or CNP.
The above natriuretic peptides and their analogues are known to be useful for treating a variety of different conditions including: edematous states such as congestive heart failure (CHF), nephrotic syndrome and hepatic cirrhosis; hypertension; pulmonary hypertension; and renal disorders as well as diseases such as renal failure due to ineffective renal perfusion or reduced glomerular filtrate rate; bacterial infections; weight loss; asthma; inflammatory-related diseases; erectile dysfunction; hypercholesterolemia; skeletal dysplasias and as a protectant for toxicity of anti-cancer drugs, as described for example in WO 2004/011498.
Typically, heart failure patients receive several chronic oral therapies, primarily including diuretics, angiotensin-converting enzyme (ACE) inhibitors, and beta-blockers. Often, these patients have an acute episode of CHF, requiring hospitalization. Following an acute episode, survivors are required to continue management of the chronic condition. The gold standard for the treatment of more advanced CHF is quadruple therapy with an ACE inhibitor (or an ARB), a beta-blocker, a diuretic and digoxin. However, each of these is associated with a number of deleterious side-effects such as neurohumoral activation, ototoxicity, hypokalemia, renal impairment and an increased risk of arrhythmic death. It has been observed that enhancement of the NP system may provide a better strategy to treat the sodium and water retention that increases cardiac preload leading to progressive cardiac dysfunction in CHF patients (Costello-Boerrigter et al., 2003, Medical Clinics of North America 87(2)). The therapeutic potential of the NPs is underscored by the role played by ANP and BNP in overcoming sodium retention in the early phase of CHF. As CHF evolves, the balance between the antinatriuretic-vasoconstrictive systems (i.e., RAAS, sympathetic nervous system, vasopressin and endothelin) and natriuretic-vasodilatory systems (i.e., NPs, nitrous oxide, prostaglandins and adrenomedulin), which has a profound effect on sodium and water homeostasis, moves towards the former system. The current hypothesis is that there is a state of relative deficiency of NP in CHF. Consequently, enhancement of NPs is considered a beneficial strategy either via administration of exogenous NPs, through agonists of NP-A, or by inhibition of the enzyme responsible for NP degradation, NEP 24.11. In tribute to the growing recognition of NP therapy, nesiritide citrate or Natrecor®, a BNP citrate salt, has been launched commercially for the intravenous treatment of acute CHF patients in the United States. A recombinant form of hANP, termed carperitide, has been launched in Japan for the same indication and is currently in clinical trials in the USA (Pharmaprojects, 2005).
An advantage of NPs over currently used diuretics is that they do not activate the RAAS system (which is associated with the progression of CHF). Furthermore, they inhibit the sympathetic nervous system (which is associated with heart failure progression, myocyte necrosis and apoptosis, and arrhythmias). It has been demonstrated that NPs exhibit fewer arrhythmic events, such as tachycardia. In trials, BNP was reported to be associated with less arrhythmic events than dobutamine (Burger et al., 2001, Am J Cardiol 88, 35-39). NPs and their derivatives can be administered alone or in combination with one or more of the following compounds: beta blockers, diuretics, ACE inhibitors, digoxin, spironolactone, anticoagulation and antiplatelet agents and angiotensin receptor blockers, as disclosed for example in WO03081246.
A disadvantage of NPs is that they are notoriously fragile in vivo, being degraded rapidly by neutral endopeptidases (NEPs) and through the clearance receptor (NPR-C), thus limiting their bioavailability. A corollary of this is that NPs, in particular Nesritide, is only useful in the treatment of acute CHF requiring intravenous infusion. To access the chronic CHF market, new stabilized NPs are sought. Such NPs have the requirements of significantly improved half lives so that more convenient routes of administration such as subcutaneous injection or oral administration can be adopted and with less frequent dosing than would be needed for current NP therapies such as ANP or BNP, both which have short half lives.
Attempts to increase resistance to proteolytic degradation of NPs through use of PEGylation and other conjugates (see, for example, WO2004/047871), resulted in improved resistance to trypsin, oral availability and in increased plasma levels as compared to native BNP in some examples. Interestingly, analysis of animal plasma assays for its lead compound reveal that their oral delivery technology is successful, in that the hBNP survives the GI tract and reaches the blood stream. However, once in the blood stream, it is degraded and cleared quite quickly (within 30 minutes the plasma levels of the compound are almost zero), demonstrating no superiority over Natrecor's 18 minute half-life. In short, hBNP is too unstable, and therefore not amenable to oral administration. An alternative approach is bioconjugation of the NP with blood components for example human serum albumin. This technology has been demonstrated to increase NP half life (see, for example, WO 2004011498). However, the corresponding large size of the resulting complex makes it unsuitable for most forms of administration. Additionally, it is not clear that the activated NP does not conjugate to other biological species in vivo with the consequence that regulatory and safety issues may arise. Other studies have targeted the stability of NPs by selecting for analogues that favor the NPR-A receptor over the NPR-C receptor (see, for example U.S. Pat. Nos. 6,028,055 and 5,846,932), thus reducing clearance mechanisms or by stabilizing the sequence against NEP protease digestion, for example by replacing the susceptible Phe with Cha. All these studies demonstrate the general amenability of NPs, without significant loss of activity, to substitution and derivatisation provided that the primary binding moieties are not disturbed.
DNP has been found to possess higher resistance to NEPs than previously known NPs, putatively due to its extended tail region. The tail region is roughly correlated to stability, with CNP, the least stable having the shortest tail. Indeed, the significantly higher potency of BNP over ANP in elevating cGMP levels in mice kidney is attributed to the greater stability of the former peptide. Correspondingly, there still exists a need for compounds with NP-like activity that are better able to withstand proteolytic enzyme attack, particularly that of NEP 24.11.
It is not possible to determine the stability of NP peptides from an examination of their primary sequences alone. The program “Peptide Cutter” available on the suite of protein analysis programs, Swissprot (http://us.expasy.org/sprot/) predicts that all known peptides have multiple regions of susceptibility to proteolytic attack. For instance, ANP is predicted to contain 59 cleavage sites for 12 enzymes, whereas BNP is predicted to contain 55 cleavage sites for a total of 13 enzymes. The peptides of the present invention are predicted to contain 59 cleavage sites for a total of 16 enzymes. Yet, as is demonstrated herein, these latter peptides are significantly more stable than their human counterparts.